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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 26-Jul-2010 to 19-Aug-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with international guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(3-aminopyrazolo[1,5-a]pyridin-2-yl)oxy]ethanol hydrochloride
EC Number:
695-745-7
Cas Number:
1079221-49-0
Molecular formula:
C9 H11 N3 O2, ClH
IUPAC Name:
2-[(3-aminopyrazolo[1,5-a]pyridin-2-yl)oxy]ethanol hydrochloride

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Based on the information provided by the Sponsor, the test item has a limited stability in aqueous solution and thus no stock solution was prepared.
Therefore, test item amounts of 5.36, 16.04, 50.23, 160.49 and 500.71 mg were weighed by means of an analytical balance and transferred to the designated test vessels with 250 mL tap water just before the start of the test. The pH was measured in each test vessel and adjusted to pH 6.0 - 6.1 with a diluted sulfuric acid or sodium hydroxide solution to improve the stability of the test item in aqueous solution.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater. The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Hardness:
/
Test temperature:
The temperature in the test media, measured in one control, was 20 °C at the start and 21 °C at the end of the incubation period.
pH:
Each test medium (test item, reference item and control) was adjusted to a pH of 6.0 - 6.2 with a diluted sodium hydroxide or sulfuric acid solution. After pH adjustment, 16 mL synthetic sewage feed was added.
Dissolved oxygen:
The concentration of dissolved oxygen did not drop below 2.5 mg/L during the incubation period.
Salinity:
/
Nominal and measured concentrations:
Nominal concentrations: 10, 32, 100, 320 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
Test item amounts of 5.36, 16.04, 50.23, 160.49 and 500.71 mg were weighed by means of an analytical balance and transferred to the designated test vessels with 250 mL tap water just before the start of the test. The pH was measured in each test vessel and adjusted to pH 6.0 - 6.1 with a diluted sulfuric acid or sodium hydroxide solution to improve the stability of the test item in aqueous solution. Immediately thereafter, 16 mL synthetic sewage feed and about 34 mL tap water were added. Finally, at the start of the test 200 mL activated sludge inoculum (adjusted to pH 6.2) was added.
Additionally, two controls, containing only tap water, synthetic sewage feed and activated sludge inoculum, were tested in parallel to the five concentrations of the test item under identical test conditions (for composition, see table on next page). Analog to the test item solution, the pH of 250 mL tap water was adjusted to pH 6.0 - 6.2 with a diluted sulfuric acid. Immediately thereafter, 16 mL synthetic sewage feed were added and the test media filled up to 300 ml with tap water. Finally, 200 mL activated sludge inoculum (adjusted to pH 6.2) was added at the test start.

TEST MEDIUM / WATER PARAMETERS
An aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 4 g dry material per liter. During the holding period of three days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge inoculum was adjusted from 7.1 to 6.2 with a diluted sulfuric acid solution.
- Synthetic sewage feed: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2 × 2H2O, 0.2 g MgSO4 × 7H2O, 2.8 g K2HPO4 filled up to a final volume of 1 liter with deionized water.
- Intervals of water quality measurement: The pH values and dissolved oxygen concentrations were determined after the addition of synthetic sewage feed and activated sludge in all test media and the controls at the start and at the end of the 3-hour incubation period. The water temperature was measured in one control at the start and at the end of the incubation period. Before the addition of activated sludge and synthetic sewage feed, the appearance of the test media was recorded.

OTHER TEST CONDITIONS
- Adjustment of pH: yes, see above
- Photoperiod: /
- Light intensity: /
- Other: During the incubation period of 3 hours all test media and the controls were continuously aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item.

EFFECT PARAMETERS MEASURED : For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated. Then the dissolved oxygen concentration was measured with an oxygen electrode (WTW TriOxmatic® 300 and an oxygen meter WTW Oxi 539, Wissenschaftlich-Technische Werkstaetten WTW, Weilheim/Germany), and was continuously recorded. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2 L-1 minute-1) was determined from the linear part of the respiration curve in the range 6.5–2.5 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of at least ten minutes.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
189 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
74 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate

Any other information on results incl. tables

Up to and including the concentration of 32 mg/L, the test item had no significant inhibitory effect (≤10%) on the respiration rate of activated sludge after the incubation period of three hours. At the higher test item concentrations of 100, 320 and 1000 mg/L, the inhibitory effects were 12, 87 and 79%, respectively. The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed only by 1% (guideline-recommendation: the two control respiration rates should be within 15% of each other).

The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 14 mg/L (the 95% confidence limits could not be calculated due to mathematical reasons). The 3-hour EC50 is within the guideline-recommended range of 5–30 mg/L, confirming suitability of the activated sludge used.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The O2 consumption rates of the 2 controls differed by 1%. The concentration of dissolved O2 did not drop below 2.5 mg/L during the incubation period. Just before measurement of the respiration rates the dissolved O2 concentrations were at least 7mg/L.
Executive summary:

The inhibitory effect of the test item on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to the EU Commission Directive 88/302/EEC, Part C.11, Commission Regulation (EC) No. 440/2008, Part C.11 and the OECD Guideline for Testing of Chemicals, No. 209.

The following nominal concentrations were tested: 10, 32, 100, 320 and 1000 mg/L.

In addition, two controls and three different concentrations of the reference item 3,5-dichlorophenol (5, 16 and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

Up to and including the concentration of 32 mg/L, the test item had no significant inhibitory effect (≤10%) on the respiration rate of activated sludge after the incubation period of three hours. At the higher test item concentrations of 100, 320 and 1000 mg/L, the inhibitory effects were 12, 87 and 79%, respectively.

The calculated 3-hour EC values were as follows :

NOEC (determined as the calculated 3-hour EC10): 74 mg/L

EC50: 189 mg/L

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